Monoclonal Antibodies
Monoclonal antibodies (Mabs) represent the fastest-growing market segment within the pharmaceutical industry. Despite a number of drawbacks, they are particularly appreciated among biotherapeutics, thanks to their unique features, including extremely high target specificity, favourable pharmacokinetics (long half-life) as well as faster development and higher success rate, as compared to small molecules. Soluble ligands and membrane-bound receptors involved in pain signalling represent ideal targets for the Mab, making it possible to obtain anti-pain biologics with higher specificity and/or different mechanism of action (MoA) as compared to currently available analgesics.
The huge body of evidence suggesting that the NGF pathway provides new molecular targets for pain therapy has spurred the development of neutralizing antibodies against NGF and its receptors. In general, antibodies in the whole immunoglobulin format (IgG) are big molecules that do not go through the blood brain barrier when they are systemically administered. As for antibodies targeting the NGF pathway this means they are likely to be effective in periphery (as it is desirable for many pain conditions) but not in central nervous system (CNS). This is a clear advantage, since NGF is known to exert neurotrophic and neuroprotective effects on specific TrkA-expressing cell populations in the CNS, like basal forebrain cholinergic neurons). From a theoretical point of view anti-NGF neutralizing antibodies represent the most efficacious tool to reduce NGF bioactivity, especially if both receptors are thought to be involved; moreover when the neutralizing agent is an antibody in the whole Immunoglobulin format (IgG), it is usually safer to block a ligand instead of a membrane-bound receptor, which would increase the risk of complement-dependent cytolysis (CDC) and antigen-dependent cell cytolysis (ADCC) for the receptor bearing cell. On the other hand, targeting NGF in Peripheral nervous System (PNS) might indirectly affect the concentration of the same factor in CNS, altering the balance between PNS and CNS pools (peripheral sink effect), with possible deleterious effects on NGF-responsive neurons in the CNS; moreover, in certain cases (e.g., the massive release of NGF in a short time, in inflammation) it is much easier to target and block the receptor than the NGF ligand. The neutralisation of either receptor would therefore avoid the drawbacks associated with NGF targeting as well as the side effects depending on signalling through the other receptor.
Various anti-TrkA antibodies have also been generated. One such antibody is the monoclonal called 5C3 as disclosed in WO97/21732. The antibody interacts within the juxtamembrane/IgG2 domain of TrkA receptor. However, this antibody was found to be a TrkA agonist and therefore not useful for inhibiting TrkA-triggered activities and consequently having a therapeutic effect, as reducing pain. As a matter of fact, when binding to TrkA, this antibody does not prevent the functional activation of the receptor, since, on the contrary, it induces the receptor functional activation upon its binding. Moreover it was not raised against specific loops in the TrkAd5 domain that are known to be essential for NGF binding.
An anti-TrkA monoclonal antibody referred to as MNAC13 is disclosed in WO00/73344. This antibody and its derivatives are said to prevent the functional activation of TrkA in a range of biological systems. In particular, the MNAC13 antibody is able to reduce pain in relevant animal models (Ugolini et al., 2007. Proc Natl Acad Sci USA. 104: 2985-2990). However, structural evidence (Covaceuszach et al., 2005. Proteins. 58: 717-727) demonstrates that the MNAC13 antibody does not interact with the NGF binding site on the TrkA receptor (TrkAd5 domain), whereas it binds to a more N-terminal portion of the TrkA extracellular domain (ECD) (i.e. the TrkAd4 domain).
WO06/131952 discloses medical uses of the MNAC13 antibody in treating chronic pain.
WO05/061540 discloses a method of antibody humanization in which structural data from crystallographic studies are employed to conduct the first design steps of the humanization process. Anti-TrkA antibodies, such as MNAC13, as disclosed in WO00/73344, are described as examples of mouse antibodies humanized using the method. Several different humanized variants of MNAC13 are disclosed in WO09/098,238.
A human antibody and derivatives thereof that acts as a powerful NGF activity antagonist by recognizing and binding specific loops in the TrkAd5 domain essential for NGF binding has not been provided yet. Moreover there is the need to obtain antibodies with a defined binding specificity to fine regulating the activity thereof.
Chronic Pain as a Therapeutic Area of Largely Unmet Need.
Persistent pain represents a major health problem. It can show different levels of severity and is associated to different pathologies, such as back injury, migraine headaches, arthritis, herpes zoster, diabetic neuropathy, temporomandibular joint syndrome, and cancer. Mild pain is presently treated with acetaminophen, aspirin, and NSAIDs. The NSAIDs inhibit COX and thereby reduce prostaglandin synthesis. However, they are associated with gastrointestinal toxicity, and although COX-2-selective inhibitors have significantly reduced adverse gastric effects, there is still a raised risk of cardiovascular disease (Zeilhofer, 2007. Biochem Pharmacol. 73: 165-174). Moderate pain can be controlled using corticosteroidal drugs such as cortisol and prednisone, inhibiting phospholipase A2 (Flower and Blackwell, 1979. Nature. 278: 456-459). Nonetheless, corticosteroids display remarkable adverse effects including weight gain, insomnia, and immune system weakening. Severe pain may be treated with strong opioids such as morphine and fentanyl. However, long-term use of opiates is limited by several serious drawbacks, including development of tolerance and physical dependence (Przewlocki and Przewlocka, 2001. Eur J Pharmacol. 429: 79-91).
As current pain therapies are often poorly effective or have undesirable side effects, an urgent need therefore exists to develop more specifically efficacious drugs directed against new molecular targets, with particular emphasis for the therapeutic area of chronic pain.
Molecular Mechanisms Underlying Pain
Chronic pain may be of either nociceptive or neuropathic origin. In some cases, complex pain syndromes are produced by a combination of both, as it is the case for many types of oncologic pain. Nociceptive pain is induced by noxious mechanical, chemical, or thermal stimuli acting through pain specific receptors, mainly expressed on the peripheral endings of sensory neurons. Activation of nociceptive Ad-fibers (small diameter, rapidly-conducting, and myelinated) and C-fibers (small diameter, slower-conducting, and unmeylinated) results in pain perception. Tonic or chronic nociceptive pain may arise from sustained inflammatory disorders, resulting in hyperalgesia (increased sensitivity to painful stimuli) and/or allodynia (lowering of the threshold beyond which a stimulus is perceived as painful). Neuropathic pain may be induced by neural lesion or dysfunction, sometimes implying central neuroplasticity.
Following injury, inflammatory mediators are released from both damaged tissues and activated immune cells. Proinflammatory cytokines such as tumor necrosis factor-a (TNFa) and interleukin-1 (IL-1) are secreted by neutrophils and activated cells of the monocyte/macrophage lineage. These cytokines may stimulate the release of Nerve growth factor (NGF) both from structural sources (fibroblasts, keratinocytes, and Schwann cells) and from inflammatory cell types (lymphocytes, macrophages, and mast cells). Moreover, mast cell degranulation releases other proinflammatory substances, which make up the so-called inflammatory soup: histamine, cytokines, prostaglandins, bradykinin, serotonin (5-hydroxytryptamine [5-HT]), adenosine triphosphate (ATP) and H+. Upon binding to their own specific receptors on nociceptive neurons, they all contribute to pain signalling (Pezet and McMahon, 2006. Annu Rev Neurosci. 29: 507-538).
TrkA Receptor Signalling in Persistent Pain.
Nerve growth factor (NGF) is a multi-functional molecule that exerts its biological functions in a variety of neural and non-neural cells (Levi-Montalcini, 1987. Science. 237: 1154-1162), by means of two types of receptors: the TrkA tyrosine kinase receptor and the p75 neurotrophin receptor (p75NTR), belonging to the molecular family of Tumor Necrosis Factor receptors. TrkA mediates the survival and neurite outgrowth-promoting effects of NGF during development. Each NGF dimer binds two TrkA monomers, resulting in dimerization and trans-autophosphorylation of specific tyrosine residues. These phosphotyrosine residues form docking sites for several adaptor proteins coupling the receptor to intracellular signalling pathways including the mitogen activated protein kinase (MAPK), phosphatidylinositol 3-kinase (PI3K) and PLC pathways (Kaplan and Miller, 2000. Curr Opin Neurobiol. 10: 381-391); (Huang and Reichardt, 2003. Annu Rev Biochem. 72: 609-642). It is known that the exogenous administration of NGF induces pain both in animals and in humans (Mantyh et al., Anesthesiology. 115: 189-204) furthermore, the first line of evidence associating NGF signalling and pain comes from genetic studies. For example, congenital insensitivity to pain with anhydrosis is an autosomal recessive disorder characterized by the absence/abnormal development of several subsets of sensory and sympathetic neurons, which makes affected individuals unresponsive to pain and unable to sweat (anhydrosis). Null mutations in the gene encoding TrkA (NTRK1) have been recognized to be responsible for this disorder (Indo, 2001. Hum Mutat. 18: 462-471; Indo et al., 2001. Hum Mutat. 18: 308-318; Indo et al., 1996. Nat. Genet. 13: 485-488).
NGF plays a key role in pain transduction mechanisms in adult nervous system. Peripheral nociceptors strongly express the TrkA and p75NTR receptors and are developmentally and functionally dependent on NGF. NGF is a peripherally produced mediator of several persistent pain states, notably those associated with inflammation, also thanks to its dual action on inflammatory mast cells that are recruited by NGF to the injured or painful site, and are induced by NGF to release inflammatory mediators. NGF is released by mast cells, fibroblasts and other cell types present in peripheral sites where inflammation is taking place. In particular, mast cells seem to play a key role (Woolf et al., 1996. J Neurosci. 16: 2716-2723). In fact, they produce NGF and display functional TrkA receptors on their surface in the same time, which makes them capable of responding to NGF itself, (Horigome et al., 1993. J Biol Chem. 268: 14881-14887). Thus the NGF-TrkA system appears to mediate mast cell activation through an autocrine loop, allowing local amplification of the activation process.
Therefore, prior art still fails to disclose an anti-TrkA molecule that specifically recognises and binds an epitope in the TrkAd5 domain, comprising the sequence from aa 294 to to aa 299 of TrkA amino acid sequence of SEQ ID NO: 65, and more effectively acts as NGF antagonists specifically preventing the functional activation of TrkA by NGF.